In this research project, we have investigated how modification of macrophages rather than modification of LDL affects macrophage metabolism of native LDL. We have learned that macrophage cholesterol accumulation can occur with native LDL when macrophages are differentiated from monocytes with the specific growth factor, M-CSF. M-CSF differentiated macrophages show constitutive uptake and degradation of native LDL. LDL uptake does not depend on macrophage oxidation of LDL or macrophage binding of LDL. Rather, the M-CSF differentiated macrophages take up LDL by constitutive fluid-phase pinocytosis, a process by which macrophages and some other cell types take up large amounts of fluid. The macrophages take up LDL as part of the fluid that they ingest during pinocytosis. This produces cholesterol accumulation in macrophages to levels characteristic of macrophage foam cells in atherosclerotic plaques. This novel mechanism of macrophage cholesterol accumulation shows that oxidative modification of LDL is not necessary for foam cell formation to occur.[unreadable] [unreadable] In related research, we have identified two distinct macrophage lineages within human coronary atherosclerotic lesions. These lineages correspond to distinct macrophage phenotypes that can be generated in vitro when human monocytes are differentiated with either GM-CSF or M-CSF. Both macrophage phenotypes are present within the human coronary artery and accumulate lipid within coronary artery atherosclerotic plaques. However, the M-CSF macrophage phenotype predominates within plaques, while the GM-CSF phenotype predominates within normal coronary artery. Thus, the M-CSF macrophage phenotype, which shows constitutive pinocytosis of LDL and is associated with inflammation in other tissues, may be an important atherogenic macrophage lineage that promotes the development of atherosclerotic plaques.[unreadable] [unreadable] In order to show that fluid-phase pinocytosis of lipoproteins such as LDL can occur within atherosclerotic lesions in vivo, it is necessary to have a tracer that does not bind cells and is similar in size to LDL. Towards this end, we have made use of fluorescent nanoparticles that do not bind cells as a tracer for qualitative and quantitative analysis of fluid-phase pinocytosis. AngioSPARK and Qtracker non-targeted Quantum Dots are stable, bright fluorophore nanoparticles designed for in vivo imaging. These nanoparticles are pegylated to minimize their interaction with cells, and remain localized in the vasculature for extended periods. Both nanoparticles are similar in size to LDL. We have shown that these nanoparticles can be used to monitor fluid-phase pinocytosis in cultured cells and by cells in vivo. Like LDL, these fluorescent nanoparticles are taken up by cultured macrophages by fluid-phase pinocytosis. Using both types of fluorescent nanoparticles, we demonstrated fluid-phase pinocytosis by macrophages in vivo in atherosclerotic lesions using the ApoE knockout mouse model of atherosclerosis. Quantum Dots were localized within atherosclerotic lesion macrophages identified with anti-CD68 antibody and foam cells identified with oil red O lipid staining. These results show that pinocytosis persists even in lipid-filled macrophage foam cells because macrophages that showed extensive lipid deposits also accumulate substantial amounts of the fluorescent nanoparticles.[unreadable] [unreadable] In conclusion, our findings show that fluid-phase pinocytosis of LDL is a plausible mechanism that can explain how macrophages accumulate cholesterol and become disease-causing foam cells. The findings direct attention to macrophage pinocytosis as a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis. Our current research is directed at identifying drugs that inhibit macrophage pinocytosis of LDL.